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Quantitative holographic agglutination assay for immunoglobulin A
Rushna Quddus1,2, Kent Kirshenbaum1, David G Grier3
1Department of Chemistry, New York University, New York, NY 10003, USA.
Insights
This study presents a novel holographic agglutination assay for quantifying immunoglobulin A (IgA) levels. This label-free, bead-free method uses Total Holographic Characterization for direct protein detection, offering a new diagnostic tool.
Area of Science:
- Biotechnology
- Analytical Chemistry
- Immunology
Background:
- Accurate quantification of immunoglobulins like IgA is crucial for diagnosing various diseases.
- Existing methods for immunoglobulin quantification can be complex, time-consuming, or require labels.
Purpose of the Study:
- To introduce a novel, label-free, and bead-free holographic agglutination assay for quantifying immunoglobulin A (IgA) in biological samples.
- To demonstrate the feasibility of using Total Holographic Characterization for direct detection of protein agglutinates.
Main Methods:
- Development of a holographic agglutination assay utilizing Jacalin lectin for selective agglutination of galactosylated immunoglobulins.
- Application of Total Holographic Characterization to analyze particle size, refractive index, and number for quantitative measurements.
- Calibration of the assay for IgA concentrations within a physiologically relevant range.
Main Results:
- The assay successfully quantified IgA in emulated biological samples with a limit of detection of 6.3 mg/dL.
- The method demonstrated reproducibility and the ability to distinguish between IgA and IgG.
- The assay covers a reference interval of IgA concentrations from 4 mg/dL to 32 mg/dL.
Conclusions:
- This study introduces a versatile platform for lectin-mediated holographic agglutination assays to monitor immunoglobulin levels.
- The developed assay offers an efficient and quantitative method for protein quantification, valuable for clinical diagnostics.
- This approach provides a foundation for developing assays for other agglutinatable proteins.
Abstract:
This study introduces a holographic agglutination assay for quantifying levels of the immunoglobulin protein IgA in biological samples. This is the first example of a label-free and bead-free assay that quantifies protein agglutinates by direct detection using Total Holographic Characterization. A proof-of-concept assay for human serum immunoglobulins is demonstrated using Jacalin, the galactose-specific plant lectin, to induce selective agglutination. By analyzing the size, refractive index, and number of particles in an assay sample, we obtain a reproducible and quantitative measurement of galactosylated immunoglobulins in a given sample. The assay is calibrated for a reference interval of IgA concentrations from 4 mg dL-1 (0.25 μM) to 32 mg dL-1 (2 μM) with a limit of detection (LOD) of 6.3 mg dL-1 (0.39 μM) in emulated biological samples. This covers the physiologically relevant concentration range with ten-fold dilution. The assay clearly distinguishes samples containing IgA from samples containing IgG. More broadly, this study introduces a platform for creating lectin-mediated holographic agglutination assays to monitor levels of immunoglobulins in biological samples. The ability to quantify immunoglobulin levels efficiently in clinical samples is likely to be valuable for diagnostics and will provide a basis for assaying other proteins that can be induced to agglutinate.
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